The relationship between cumulative dose of immunosuppressive agents and COVID‐19‐associated mucormycosis: A multicenter cross‐sectional study

Abstract Background and Aims Immunosuppressive therapy has a key role in developing coronavirus disease‐2019 (COVID‐19)‐associated mucormycosis. In this study, we investigated the effect of the type and cumulative dose of immunosuppressive agents on COVID‐19‐associated mucormycosis. Methods We designed a descriptive cross‐sectional study involving three COVID‐19 hospitals in Iran. Clinical and demographic data were gathered from the medical records and checked by two independent researchers to minimize errors in data collection. Results Seventy‐three patients were included in the study. The mean age of cases was 57.41 (SD = 12.64) and 43.8% were female. Among patients, 20.5% were admitted to the intensive care unit (ICU) during COVID‐19. Furthermore, 17 patients (23.29%) had a history of diabetes mellitus. Sixty‐nine patients (94.52%) had a history of receiving corticosteroids (dexamethasone) during treatment of COVID‐19, and of those, five patients (6.85%) received Tocilizumab beside. The mean cumulative dose of corticosteroids prescribed was 185.22 mg (SD = 114.738). The average cumulative dosage of tocilizumab was 720 mg (SD = 178.89). All of the included patients received amphotericin B for mucormycosis treatment, and 42 survived (57.53%). Also, there was a significant relationship between hospitalization in ICU for COVID‐19 and the mucormycosis outcome (p = 0.007). However, there weren't any significant associations between cumulative doses of immunosuppressive drugs and mucormycosis outcome (p = 0.52). Conclusion The prevalence of COVID‐19‐associated mucormycosis is increasing and should be considered in the treatment protocols of COVID‐19. Controlling risk factors such as diabetes, malignancy and the administration of immunosuppressive agents based on recommended dosage in validated guidelines are ways to prevent mucormycosis.

affecting the incidence and control of mucormycosis owns of great importance.
Immunosuppressive therapy is one of the risk factors that could be crucial in developing COVID-19-associated mucormycosis. In the procedure of COVID-19 treatment, a variety of immunosuppressive medications are used in varying doses to reduce the symptoms of the host immune response and cytokine storm. 7 This includes a number of medications such as corticosteroids, baricitinib, and tocilizumab (Actemra). Thus, by examining the impact of different medications and doses used to suppress the immune system on the risk of developing mucormycosis, immunosuppressive medications in COVID-19 patients might be prescribed with greater prudence. Therefore, in this study, we investigated the effect of the type and cumulative dose of immunosuppressive agents on COVID-19associated mucormycosis.

| Study design
We performed a descriptive cross-sectional study involving three COVID- 19

| Study subjects and definitions
Patients were included in the study based on the following criteria: Institutes of Health (NIH) guidelines for COVID- 19. 8 Clinical and demographic data were gathered from the medical records and checked by two independent researchers to minimize errors in data collection. Patients were informed that their demographic and clinical information would be used in the study, and written informed consent was obtained. The cumulative dose of immunosuppressive therapy was defined as the total dose of any immunosuppressive drug received for the treatment of COVID-19.

| Statistical methods
Data were gathered into an Excel spreadsheet and then transferred to SPSS Statistics 26.0 (IBM, Inc). Descriptive analysis was performed, including frequencies, mean, and standard deviation (SD). Also, bivariate analysis was conducted with the Chi-square test and independent-sample T-test.

| RESULTS
Initially, 111 patients with COVID-19-associated mucormycosis were included in the study; however, owing to the unavailability of data on the course of COVID-19; finally, 73 patients were included in the study. The mean age of cases was 57.41 ± 12.64% and 43.8% were female. Among patients included in the study, 20.5% were admitted to the intensive care unit (ICU) during COVID-19. Furthermore, 17 patients had a history of diabetes mellitus. Sixty-nine patients had a history of receiving corticosteroids (dexamethasone) during treatment of COVID-19, and of those, five patients received Tocilizumab beside. The mean cumulative dose of corticosteroids prescribed was 185.22 mg ± 114.738. The average cumulative dosage of Tocilizumab was 720 mg ± 178.89. All of the included patients received amphotericin B for mucormycosis treatment, and 42 survived. Table 1 shows the summary of the characteristics of patients. In addition, the demographic and clinical characteristics of patients based on mucormycosis outcome were compared in Table 2, which indicates a significant relationship between hospitalization in ICU for COVID-19 and the mucormycosis outcome (p = 0.007). However, there weren't any significant differences in cumulative doses between two groups of survivors and non-survivors of mucormycosis (Table 3).

| DISCUSSION
As of the time of writing this article, COVID-19 has infected more than 500 million people and caused the death of about 6 million patients. 9 Among hospitalized COVID-19 patients, mucormycosis, as a secondary infection, has had a prevalence of seven cases per 1000 patients which is 50 times higher than its prevalence among diabetic patients (0.14 cases per 1000). 10 The mortality rate of mucormycosis is about 46%, and a delay in diagnosis only for a week will increase the risk of death from 35% to 66%, which can be prevented by timely diagnosis. 11,12 In our study, 42.5% of patients with mucormycosis lost their lives, demonstrating a high risk of death in COVID-19-associated mucormycosis. In addition, 18 individuals had a history of underlying conditions such as diabetes mellitus and malignancy. As a result, managing underlying diseases could be an essential factor in reducing the risk of mucormycosis. Another important point is the type of drug used to suppress the immune system. In our study, all people who received immunosuppressive therapy took dexamethasone as a corticosteroid, which could be due to its lower cost, availability, and better efficacy. 14   In COVID-19, there is a significant reduction in the number of clusters of differentiation 4 (CD4) and CD8 cells. 18 Therefore, this immunosuppression could increase the risk of secondary infections.
Previous studies have reported that a significant result of hyperglycemia is increased expression of glucose-regulated protein (GRP78) expression. This protein is located in the endoplasmic reticulum of mammalian cells and could act as a receptor for Rhizopus spore coat protein homologs (CotH) protein kinase. Thus, it facilitates the attachment of the fungus particle to endothelial cells. 19 The key point of this finding is that the expression of GRP78 in COVID-19 patients has increased compared to controls, which may indicate that the SARS-CoV-2 virus, regardless of the immunosuppressive therapy, can be a risk factor for developing mucormycosis. 20 Another finding of our study was the association between the severity of COVID-19 disease and the outcome of mucormycosis.
Due to the limited available information, the severity of COVID-19 was considered based on hospitalization in the ICU, which shows an increased likelihood of severe mucormycosis and death outcomes.
However, this finding may indicate that people admitted to the ICU for COVID-19 have certain risk factors that may affect the outcome of mucormycosis, which might be due to higher doses of immunosuppression, although it was not evaluated in this study.
The incompleteness of data in medical records and lack of microbiological diagnosis of mucormycosis were our main limitations.
Furthermore, the limited use of non-corticosteroid immuno- writingreview & editing.

ACKNOWLEDGMENTS
All affiliations with or financial involvement in any entity with a financial interest in, or in competition with, the manuscript's subject matter are disclosed. This includes stock ownership, employment, consultancies, honoraria, grants, patents, and royalties. This research did not receive any specific assistance from funding agencies in the public, commercial, or not-for-profit sectors.

CONFLICTS OF INTEREST
The authors declare no conflicts of interest.

CONSENT FOR PUBLICATION
All authors have read and approved the final version of the manuscript corresponding author had full access to all of the data in this study and takes complete responsibility for the integrity of the data and the accuracy of the data analysis. This article has not been published and is not under consideration for publication elsewhere.

DATA AVAILABILITY STATEMENT
All information provided in this article can be obtained from the author on request.

ETHICS STATEMENT
We also declare that the study was performed according to international, national, and institutional rules considering animal experiments, clinical studies, and biodiversity rights.

TRANSPARENCY STATEMENT
The lead author Ali Asadollahi-Amin affirms that this manuscript is an honest, accurate, and transparent account of the study being reported; that no important aspects of the study have been omitted; and that any discrepancies from the study as planned (and, if relevant, registered) have been explained.